Graft-versus-host disease (GVHD) is one of the major causes of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). GVHD is mediated by donor T-cells that recognize minor or major MHC disparities in the recipient. Recently, our laboratory and other groups have shown that CD4+CD25+regulatory T cells (Tregs) when infused with allogeneic effector T-cells can block tissue damage and improve survival. The process by which Tregs inhibit effector cell function during GVHD is incompletely understood. The critical site at which this occurs is not clear, the mechanisms by which Tregs migrate into lymphoid tissue is only partly understood, and whether Treg migration to target organs such as the gastrointestinal tract is important in mitigating GVHD has not been explored. The principal goal of this project will be to examine the role of the chemokine receptors CCR4 and CCR7 in the migration of Treg cells during GVHD. To accomplish this, we will use CCR4 and CCR7 knockout mice extensively backcrossed onto a C57BL/6 background. We will isolate CD4+CD25+ Tregs from knockout and wild-type animals, and compare their ability to protect against GVHD when transferred into haplo-identical recipients. In the case of CCR4, we will focus specifically but not exclusively on the role of Treg migration to the skin and gut in preventing complications at these sites and improving overall recipient survival. We will also focus on the hypothesis that CCR7 is required for naive Treg migration into recipient lymph nodes at the time of transplantation, and that this critical step is necessary for previously unstimulated Tregs to protect against GVHD lethality. To evaluate the trafficking of Treg cells we will use flow cytometry and our pioneering work with in-vivo stereofluorescence microscopy using eGFP transgenic wild type, CCR4 -/-, and CCR7-/- animals. The goal of this work is to identify potential targets for the prevention and treatment of GVHD.